A bacterial infection is one of the most common and fatal cause to the human diseases, unfortunately, an abuse of the antibiotics has caused an antibiotics resistance to bacterium. In fact, the resistance rate of bacterium to a new antibiotics is much faster than the developing rate of newly made antibiotics analogue. For example, Enterococcus faecalis, Mycobacterium tuberculosis and Pseudomonas aeruginosa, which can be threaten to a life, have grown the resistance to all antibiotics known up to the present (Stuart B. Levy, Scientific American, 46-53, 1998).
The tolerance to the antibiotics is a distinguished phenomenon from the resistance to the antibiotics. The above tolerance to the antibiotics was firstly found from Pneumococcus sp in the 1970's and gave an important clue to a mechanism of action of Penicillin (Tomasz et al., Nature, 227, 138-140, 1970). The species having the tolerance to the antibiotics stop growing under the normal concentration of the antibiotics, but do not die in the event. The tolerance is caused because an activity of the autolytic enzymes, like autolysin, is not occurred when the antibiotics inhibits a cell wall's synthetase. In case of Penicillin, it may kill a bacterium by activating an endogenous hydrolytic enzymes, but in another case a bacterium may be survived at the time of antibiotics' treating by controlling an activation of the enzyme.
Having the tolerance to a bacterium is clinically very important this is because if it is impossible to kill the tolerance bacterium, the effective of antibiotic's treating to a clinical infection may be decreased (Handwerger and Tomasz, Rev. Infec. Dis., 7, 368-386, 1985). In addition, the tolerance is regarded as a kind of essential prerequisite to generate the bacterium's resistance to the antibiotics as there may be certain survived strain by the antibiotic treating. The survived strain can grow continually under the existence of the antibiotics by way of obtaining a new genetic element having the resistance to the antibiotics. In fact, it is known that all the bacterium having the resistance to the antibiotics also have the tolerance to the antibiotics (Liu and Tomasz, J. Infect. Dis., 152, 365-372, 1985), thus it is necessary to develop the new antibiotics, which can kill a bacterium having the resistant to the antibiotics.
The tolerance can be divided into two cases in a point of a mechanism of action, wherein the first case is a phenotypic tolerance, which is generated from all the bacterium when the growth rate is decreased (Tuomanen E., Revs. Infect. Dis., 3, S279-S291, 1986) and the second case is a genetic tolerance by a mutation, which is generated from a certain bacterium. A basic phenomenon for both cases is the regulation of decreasing the activation of autolysin enzyme. This regulation may be temporary when it is the phenotypic tolerance by an external stimulus, but the regulation may be permanent when it is the genetic tolerance causing the change of channel for regulating a hemolysis. Evidently, the simplest genetic tolerance is the one generated by the lack of autolysin enzyme. However, due to several uncertain reasons, there has been no precedent case of clinically finding the strain having the tolerance by the lack of autolysis enzyme, preferably the tolerance found clinically is made under the process of regulating the activation of autolysin enzyme (Tuomanen et al., J. infect. Dis., 158, 36-43, 1988).
As examined in the above, the development of the new antibiotics is needed in order to kill the bacterium having the resistance to the antibiotics and it is necessary to develop the new antibiotics, which can act independently irrespective of the activation of the autolysin enzyme.
Meanwhile, the bacterium can kill a neighboring bacterium by synthesizing peptides named as a bacteriocin or small organic molecules, wherein those bacteriocins are structurally divided into three kinds. The first kind is lantibiotics, the second kind is nonlantibiotics, and the third kind is the one secreted by a signal peptide (Cintas et al., J. Bad., 180, 1988-1994, 1998). The animal, including the insect, can also produce a peptide antibiotics by themselves (Bevins et al., Ann. Rev. Biochem., 59, 395-414, 1990), wherein there may be three divided groups according to the structure. The first group is a cysteine-rich β-sheet peptide, second group is an α-helical amphiphilic peptide molecule, and third group is a praline-rich peptide (Mayasaki et al., Int. J. Antimicrob. Agents, 9, 269-280, 1998). It is well known that these kinds of antibacterial peptides play an important role both in host defense and innate immune system (Boman, H. G., Cell, 65:205, 1991; Boman, H. G., Annu. Rev. Microbiol., 13:61, 1995). Additionally, the antibacterial peptides have various structures according to the amino acid sequence. The most common one among the structures is the structure forming an amphiphilic alpha helical structure but without cysteine residue like a cecropin, which is the antibacterial peptide, found in the insect.
Although there has been a hypothesis that a peptic ulcer is caused by a stress and a product of hyperchylia, however interest is on a Helicobacter pylori bacterium after it is disclosed that the peptic ulcer is caused by the Helicobacter pylori bacterium (Blaser, M J., Trends Microbiol., 1, 255-260, 1991). The Helicobacter pylori bacterium belonging to the Gram negative bacterium is very slow in the growth rate and is anaerobic microorganism having a helical body and flagella. RPPL1 protein among the most proteins produced by the Helicobacter pylori bacterium is consisted of 230 numbers of amino acid and it is disclosed that the amino terminal of the proteins has the same structure as the cecropin's, especially eight number of amino acid. The RPL1's amino terminal of the Helicobacter pylori bacterium has a complete amphiphilic helical-shaped structure (Putsep, K. et al., Nature, 398, 671-672, 1999). There has been a report about the mechanism of action that the amphiphilic peptide destructs the lipid of the microorganism by connecting with the lipid of cell membrane of the microorganism or by changing a displacement of the lipid of cell membrane because the amphiphilic peptide is comprised of the structure similar to the lipid of cell membrane. In addition, there has been a report that the amino terminal of RPL1's protein in the Helicobacter pylori bacterium also has the antibacterial activity (Putsep K. et al., Nature, 398, 671-672, 1999).
Accordingly, a lot of researches have been made, and using these researches, lots of researches to develop the antibiotics to the bacterium have also been tried. The amphiphilic peptides being reported until now are HP (2-20) peptide and melittin peptide and etc.
It has been reported that HP (2-20) peptide, which has the amphiphilic activation among the parts of the amino terminal of RPL1's protein derived from the Helicobacter pylori along with having the antibiotic activation, has not a cytotoxicity but has antibacterial activation together with an antifungal effect (Biochem. Biophys. Res. Commun., 2002, 291, 1006-1013, Biochim. Biophys. Acta. 2002, 1598, 185-194).
Besides, it has been reported that the melittin peptide, which occupies more than 50% of the Pan-Cake among the bee venom's ingredients, wherein a carboxy terminal has become amidation. And it has been reported that the melittin peptide can destruct the cell of the higher animal well under the low concentration due to having the high cytotoxicity to a eukaryotic cell and has the antibacterial activation to the Gram negative bacterium and Gram positive bacterium (Habermann, E., Science, 177: 314, 1972; Steiner, H., et al., Nature, 292: 246, 1981; Tosteson, M. T., et al., Biochemistry, 228: 337, 1987).
What is more, the amphiphilic peptide belonging to the cecropin series HP (2-20) comprising the amino acid similar to the HP (2-20) was firstly found from a drosophila, and since then it is also found from a silk worm pupa and a small intestine of a pig. Among them, it has been reported that a cecropin A has the high antibacterial activation, but has the low antifungal and anticancer effect (Boman, H. G. and Hultmark, D., Annu. Rev. Microbiol., 41: 103, 1987).
Also, in addition to the research about the activation of the above amphiphilic peptide, it is confirmed that the characteristic of sequence is closely related to the antibacterial activation when inspecting the amino acid sequence and protein structure of the amphiphilic peptide. Therefore, a conjugation peptide can be made by substituting the certain parts of the sequence with the similar amino acid using the amino acid sequence of the above amphiphilic peptide or by recombinating the certain sequences. And the production of a new synthetic peptide having the excellent antibacterial, antifungal or anticancer activation can be also made by inversing the certain parts of the function of the peptide sequence (Chan, H. C., et al., FEBS Lett., 259: 103, 1989; Wade, D., et al., Int. J. Pept. Prot. Res., 40: 429, 1992).
In fact, a synthetic peptide mag A and mag G, which have the anticancer effect, were prepared by applying the amphiphilic peptide and the potency was also reported (Ohsaki, et al., Cancer Res., 52: 3534, 1992). Additionally, the synthetic peptides having the antifungal activation by mutually connecting the amino acids in the amphiphilic parts, flexibility parts and hydrophobic parts from a magainin 2 and melitin peptides have been developed, and those developed peptides was granted as a patent because of the action to bacteria and the strain in a fungus (KR Patent no. 0204501). Also, the inventors for the present invention substituted the certain amino acids of the existing HP (2-20) peptide with a tryptophan and resulted in the addition of hydrophobic (sequence no. 2) By doing so, the inventors confirmed the addition of the antibiotics effect and the present invention was granted a patent with the antibiotics peptide (KR Patent no. 0459808). Also, the present inventors synthesized the antibiotics peptide, which was only left the helical structure of the peptide but added the cation property, by amputating the relaxation structure from the antibiotic peptide comprising of the helical structure in a straight line, wherein they confirmed the high effect of the antibacterial and antifungal of the above peptide without having the cell toxicity and filed an application with the above contents (KR Patent no. 10-2007-0088127).
Recently, lots of researches to develop an excellent antibiotic peptide having more antibacterial activity and less cell toxicity than the existing antibiotic peptides.
Accordingly, the present inventors have tried to develop the excellent antibiotic peptide using the existing antibiotic peptide. By the process of development try, they have completed the present invention by confirming the fact that both the new peptide comprising of amino acid sequence of SEQ. ID. NO:2, which was produced by substituting both the first and the eighth position of Phenylalanine from the antibiotic peptide each alanine comprising the amino acid sequence of the existing SEQ. ID. NO:1, and the new peptide comprising the amino acid sequence of SEQ. ID. NO:3, which was produced by substituting Asparagine at the thirteenth position of the above peptide with lysine have less cell toxicity and have similar antibacterial activity or more antibacterial activity than the antibiotic peptide comprising of amino acid sequence of SEQ. ID. NO: 1